The spindle checkpoint is a cell-cycle surveillance system that prevents premature sister-chromatid separation in mitosis and ensures the accuracy of chromosome inheritance. A multisubunit ubiquitin ligase complex called the anaphase-promoting complex or cyclosome (APC/C) is required for sister-chromatid separation. The spindle checkpoint protein Mad2 binds to Cdc20, the mitotic activator of APC/C, and inhibits APC/CCdc20, thus delaying the onset of anaphase. Mad2 is positively regulated by Mad1 and inhibited by p31comet. In mitosis, the Mad1-Mad2 core complex recruits cytosolic Mad2 to kinetochores through Mad2 conformational dimerization and converts Mad2 to an intermediate conformer (I-Mad2) more amenable to Cdc20 binding, thus facilitating checkpoint activation. During checkpoint inactivation, p31comet binds to Mad1- or Cdc20-bound Mad2, thereby preventing Mad2 activation and promoting Cdc20 autoubiquitination and the dissociation of Mad2 from Cdc20. We have previously determined the structures of both latent and active conformers of human Mad2 using nuclear magnetic resonance (NMR) spectroscopy. In unpublished preliminary results, we have determined the crystal structures of the symmetric Mad2 dimer and the Mad2-p31comet complex. These structures have provided key insights into Mad2 regulation. In this proposal, we will further investigate the regulation of Mad2 by Mad1 and p31comet. In Aim 1, we will determine the structure of intermediate Mad2 (I-Mad2) by NMR. In Aim 2, we will characterize the structure and function of the C-terminal domain (CTD) of Mad1. In Aim 3, we will perform both structural and biochemical analysis of the p31comet-Mad2-Cdc20 complex. Defects of the spindle checkpoint cause aneuploidy, which is a prevalent form of genomic instability in human cancers. The proposed research will shed light on the molecular mechanism of the spindle checkpoint and help us understand the root causes of aneuploidy. PUBLIC HEALTH RELEVANCE: In this proposal, we will further investigate the regulation of spindle checkpoint Mad2 by Mad1 and p31comet with a combination of structural, biochemical and cell biological approaches. Defects of the spindle checkpoint cause aneuploidy (abnormal numbers of chromosomes), which is a prevalent form of genomic instability in human cancers. The proposed research will shed light on the molecular mechanism of the spindle checkpoint and help us understand the root causes of aneuploidy.